Strain belonging to bacillus genus, microbiological agent, and plant cultivation method

ABSTRACT

The present invention relates to the  Bacillus  sp. strains AT-332 (NITE BP-1095) and AT-79 (NITE BP-1094) isolated from nature; and a plant disease control agent, a nematode control agent and a plant growth promoter containing the strains as active bacteria. The  Bacillus  sp. strains AT-332 and AT-79 strain are effective in controlling both a wide range of various plant diseases and nematode-damage and capable of promoting the growth of useful plants, due to a culture containing a secondary metabolite of the strains or cultivated and isolated live bacteria of the strains being introduced to a plant body or to the culture soil.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/119,022, filed Nov. 20, 2013, which is a National Stage ofInternational Application No. PCT/JP2012/062935, filed May 21, 2012,claiming priority based on International Patent Application No.PCT/JP2011/062109, filed May 26, 2011, the contents of all of which areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a novel microorganism useful forcontrolling plant diseases and nematode damage and promoting the growthof plants. Specifically, the present invention relates to the Bacillussp. strains AT-332 and AT-79, which are a novel microorganism exhibitingmuch superior effects in controlling plant diseases and nematode damageand promoting the growth of plants compared to microorganisms belongingto a closely-related Bacillus amyloliquefaciens disclosed in theliterature; and a plant disease control agent, a nematode control agentand a plant growth promoter containing the fungus body and the cultureof the microorganisms.

BACKGROUND ART

A main method for controlling plant diseases and nematodes is a methodusing chemical pesticides, and chemical pesticides have enabled stableproduction of crops to date. However, recently, it has become difficultto fully control the impact on the environment due to continuous use ofchemical pesticides and emergence of drug-resistant bacteria by theconventional chemical pesticides; and diseases such as a bacterialdisease which are difficult to control are developing into a majorproblem. Accordingly, biological control technology using amicroorganism isolated from nature draws increasing attention and someof microorganism pesticides have been commercially-produced. However,the conventional microbiological pesticides have a defect that theeffect is not stable and applicable diseases are fewer compared tochemical pesticides. In these circumstances, there has been growingdemand for a novel microbiological pesticide which has new applicablediseases and exhibits a stable control effect.

As a plant disease control agent using a microorganism, a Talaromycesflavus agent, a Pseudomonas fluorescens agent, an avilurence Erwiniacarotovora agent, a Trichoderma atroiviride agent, a Bacillus simplexagent, a Bacillus subtilis agent and the like are registered as amicrobiological pesticide and have been used.

As a nematode control agent using a microorganism, a Pasteuria penetransagent and a Monacrosporium phymatophagum agent are registered as amicrobiological pesticide and have been used.

The specification of Japan Patent No. 2955655 (Patent Document 1)discloses a plant disease control agent using bacteria belonging toBacillus amyloliquefaciens. The active ingredient of the plant diseasecontrol agent is the product of the microorganism and the bacteria perse are not used as a pesticide. Furthermore, the control target is adisease caused by filamentous bacteria and the document does notdisclose the control of the bacterial disease. JP-A-2009-247302publication (Patent Document 2) discloses a microorganism pesticidewhich can control the disease by filamentous bacteria and the bacterialdisease at the same time in which viable bacteria cells per se areeffective, but the document has no description on the nematode control.

The specification of Japan Patent No. 3471815 (Patent Document 3; WO98/050422) discloses a plant disease control agent using Bacillusbacteria which can be used for a wide range of plant diseases andeffective on corn rootworms, but the document has no description on thenematode control. The specification of Japan Patent No. 4071036 (PatentDocument 4; US 2004/265292) discloses the Bacillus sp. D747 strain whichcan be used for controlling plant diseases and harmful insects, but thedocument has no description on the nematode control.

The specification of Japan Patent No. 3471811 (Patent Document 5; WO96/032840) discloses a nematode control agent using Bacillus genusbacteria. The active ingredient of the nematode control agent is thebacteria or spore of the Bacillus firmus strain having an antinematodeactivity but the document has no description on the plant diseasecontrol. The specification of Japan Patent No. 4359653 (Patent Document6; WO 1997/012980) discloses a method for controlling nematodes using atoxin produced by a novel Bacillus thuringiensis strain but the documenthas no description on the plant disease control.

In agriculture, chemical fertilizers are an important agriculturalmaterial which influences the yield of crops. However, 30 to 50% of theused chemical fertilizer components are not utilized in the crops butdiffused in the environment, which causes eutrophication of rivers andgroundwater contamination. A large quantity of fossil fuels is used inthe production of chemical fertilizers and the production cost of thechemical fertilizers is increasing along with the soaring prices offossil fuels. Furthermore, nitrogen oxide (NOx) as a decompositionproduct of a nitrogen fertilizer is said to be about 300 times moreefficient in greenhouse emissions than carbon dioxide, and there isgrowing concern about global warming. Food shortage is expected infuture due to the global population growth and therefore use of amaterial in order to increase the crop productivity is inevitable andthere is growing need for a more environmentally-friendly material toreplace the conventional chemical fertilizers.

In the light of such circumstances, studies have been made mainly on abroad range of Rhizobium bacteria, Pseudomonas bacteria and Bacillusbacteria. However, very few are in practical use because they are lesseffective.

As discussed above, no Bacillus bacterium which is effective on plantdiseases in general, available in controlling nematodes and is effectivein promoting plant growth has been known to date.

PRIOR ART DOCUMENTS Patent Documents

-   Patent Document 1: Japan Patent No. 2955655-   Patent Document 2: JP-A-2009-247302-   Patent Document 3: Japan Patent No. 3471815-   Patent Document 4: Japan Patent No. 4071036-   Patent Document 5: Japan Patent No. 3471811-   Patent Document 6: Japan Patent No. 4359653

DISCLOSURE OF INVENTION Problem to be Solved by the Invention

An object of the present invention is to isolate a novel microorganismfrom nature to provide, which microorganism has effects of controllingmultiple plant diseases, controlling nematodes and/or promoting plantgrowth.

Another object of the present invention is to provide a plant diseasecontrol agent, a nematode control agent and a plant growth promoter,which contain the above-mentioned microorganism as active bacteria andcan be used as a biological pesticide (microbiological agent).

Means to Solve the Problem

As a result of intensive studies to solve the problem, the presentinventors have succeeded in isolating a novel strain belonging toBacillus genus from nature, which strain has effects of controllingmultiple plant diseases, controlling nematodes and promoting plantgrowth, and accomplished the present invention.

The present invention relates to the strain described in 1 to 4 below,the microbiological agent in 5 to 8 below and the method for cultivatingplants in 9 below.

1. A strain comprising 16S rDNA represented by the base sequence No. 2or 3.2. The strain as described in 1 above, wherein the strain per se and/orthe culture of the strain shows effects of controlling plant diseases,controlling nematodes and/or promoting plant growth.3. The Bacillus sp. AT-332 strain as described in 1 or 2 above,containing 16S rDNA represented by the base sequence No. 2.4. The Bacillus sp. AT-79 strain as described in 1 or 2 above,containing 16S rDNA represented by the base sequence No. 3.5. A microbiological agent containing the strain and/or the culture ofthe strain described in any one of 1 to 4 above as an active ingredient.6. The microbiological agent as described in 5 above, which is a plantdisease control agent.7. The microbiological agent as described in 5 above, which is anematode control agent.8. The microbiological agent as described in 5 above, which is a plantgrowth promoter.9. A method for cultivating plants, treating the plants with themicrobiological agent described in any one of 5 to 8 above.

Effects of the Invention

The Bacillus sp. strains AT-332 and AT-79 of the present invention cancontrol a wide range of various plant diseases and nematodes andfurther, can promote the growth of useful plants due to the culture(including viable bacteria cells) or cultivated and isolated livebacteria of the strains being introduced to a plant body such as roots,stems, leaves, seeds and fruits or to the culture soil.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows the molecular phylogenetic tree using 16S rDNA basesequence of Bacillus sp. strains AT-332 and AT-79. In the figure, thenumbers near the branches are the bootstrap values and a scale bar isshown at the lower left.

FIG. 2 Photographs (a) to (d) show the effect of promoting plant growthof the AT-332 strain in the basic test (Example 12 and ComparativeExamples 12-13).

FIG. 3 shows the effect of promoting growth of Chinese cabbage of theAT-332 and AT-79 strains in a pot test (Example 13).

MODE FOR CARRYING OUT THE INVENTION

The present inventors screened for microorganisms from various plants,soils and the like for the purpose of newly developing a safe andsuperior microbial pesticide and/or microbial fertilizer which have abroad antibacterial spectrum against various plant diseases, showantinematode activity and have effect of promoting plant growth. As aresult, the present inventors have made a useful finding that the strainisolated from the soil collected in Ibaraki Prefecture shows a broadantibacterial spectrum against various plant diseases, shows highinsecticidal activity against nematodes and has effect of promotingplant growth.

The thus-newly-isolated both strains (AT-332 strain and AT-79 strain)are a gram-positive motile bacillus as is clear from the bacteriologicalcharacteristics to be described later, and grow and form spores under anaerobic condition. The both strains turned out positive in both ofcatalase reaction and oxidase reaction. Furthermore, as a result ofidentification based on the about 1500 bp-base sequence from the 5′terminal side of 16S rDNA, the strains were confirmed to be a novelstrain belonging to bacillus genus related to Bacillusamyloliquefaciens. Due to the superior characteristics of having effectson a wide range of plant diseases, high control effect on nematodes andeffect of promoting plant growth, the AT-332 and AT-79 strains wereidentified as a novel strain and designated as the Bacillus sp. AT-332and AT-79 strains related to Bacillus amyloliquefaciens.

Bacillus sp. AT-332 strain and AT-79 strain of the present inventionhave been deposited as Bacillus sp. AT-332 and Bacillus sp. AT-79 strainwith the depositary institution, Biological Resource Center, NationalInstitute of Technology and Evaluation (2-5-8 Kazusakamatari,Kisarazu-shi, Chiba 292-0818 JAPAN) (original deposit date (accepteddate): May 2, 2011; Accession number: NITE BP-1095 and NITE BP-1094).

The bacteriological characteristics of Bacillus sp. AT-332 (NITEBP-1095) are described below. The bacteriological characteristics havebeen determined in reference to the following documents. PRIEST (F. G.),GOODFELLOW (M.), SHUTE (L. A.) and BERKELEY (R. C. W.): Bacillusamyloliquefaciens sp. nov., nom. rev. Int. J. Syst. Bacteriol., 1987,37, 69-71 and Bergey's Manual of Systematic Bacteriology, Second Editionvolume 3.

(1) Morphological Property

-   Form: rod-shaped bacterium-   Size: width of 0.8 to 0.9 μm and length of 1.5 to 2.0 μm-   Mobility: +-   Epiphytic state of flagellum: peritrichous-   Presence or absence of spores: +(quasi-terminal)

(2) Cultural Characteristics

-   Culture medium: nutrient agar medium (30° C.)-   Form: circular-   Prominence: flat-   Periphery: entire margin-   Surface status: smooth-   Viscosity: viscous-   Transparency: opaque-   Color hue: cream color-   Gloss: dull-   Pigment production: non-productive

(3) Physiological Characteristics

-   Gram staining: +-   Nitrate reduction: −-   Nitrogen desorption reaction: −-   MR test: −-   VP test: +-   Indole generation: −-   Hydrogen sulfide generation: −-   Hydrolysis of starch: +-   Use of citric acid: −(Koser)    -   +(Christensen)-   Use of inorganic nitrogen source: −(nitrate)    -   +(ammonium salt)-   Urease: −-   Oxidase: +-   Catalase: +-   Range for growth pH 5: +    -   pH 8: +    -   pH 9: +-   Temperature for growth 37° C.: +    -   45° C.: +    -   50° C.: +    -   55° C.: −-   Growth in anaerobic condition: −-   OF test (oxidation/fermentation): −/−-   Acid production/gas production from sugars:    -   L-arabinose: +/−    -   D-glucose: +/−    -   D-fructose: +/−    -   Maltose: +/−    -   Lactose: −/−    -   D-sorbitose: +/−    -   Inositol: +/−    -   D-xylose: +/−    -   D-mannose: +/−    -   D-galactose: −/−    -   Saccharose: +/−    -   Trehalose: +/−    -   D-mannnitole: +/−    -   Glycerin: +/−-   β-galactosidase activity: −-   Arginine dihydrolase activity: −-   Lysine decarboxylase activity: −-   Tryptophan deaminase activity: −-   Gelatinase activity: +

The bacteriological characteristics of Bacillus sp. AT-79 (NITE BP-1094)are described below.

(1) Morphological Property

-   Form: rod-shaped bacterium-   Size: width of 0.8 to 0.9 μm and length of 1.5 to 2.0 μm-   Mobility: +-   Epiphytic state of flagellum: peritrichous-   Presence or absence of spores: +(quasi-terminal)

(2) Cultural Characteristics

-   Culture medium: nutrient agar medium (30° C.)-   Form: circular-   Prominence: flat-   Periphery: entire margin-   Surface status: smooth-   Viscosity: viscous-   Transparency: opaque-   Color hue: cream color-   Gloss: dull-   Pigment production: non-productive

(3) Physiological Characteristics

-   Gram staining: +-   Nitrate reduction: −-   Nitrogen desorption reaction: −-   MR test: −-   VP test: +-   Indole generation: −-   Hydrogen sulfide generation: −-   Hydrolysis of starch: +-   Use of citric acid: −(Koher)    -   +(Christensen)-   Use of inorganic nitrogen source: −(nitrate)    -   +(ammonium salt)-   Urease: −-   Oxidase: +-   Catalase: +-   Range for growth pH 5: +    -   pH 8: +    -   pH 9: +-   Temperature for growth 37° C.: +    -   45° C.: +    -   50° C.: +-   Growth in anaerobic condition: −-   OF test (oxidation/fermentation): −/−-   Acid production/gas production from sugars:    -   L-arabinose: +/−    -   D-glucose: +/−    -   D-fructose: +/−    -   Maltose: +/−    -   Lactose: −/−    -   D-sorbitose: +/−    -   Inositol: +/−    -   D-xylose: +/−    -   D-mannose: +/−    -   D-galactose: −/−    -   Saccharose: +/−    -   Trehalose: +/−    -   D-mannnitole: +/−    -   Glycerin: +/−-   β-galactosidase activity: −-   Arginine dihydrolase activity: −-   Lysine decarboxylase activity: −-   Tryptophan deaminase activity: −-   Gelatinase activity: +

The base sequences from the 5′ terminal side of 16S rDNA of the Bacillussp. AT-332 strain and AT-79 strain of the present invention arerepresented by sequence No. 2 and sequence No. 3, respectively.

Sequence No. 2 and sequence No. 3 differ from each other only in twobases at base No. 444 and base No. 1242. Base No. 444 is guanine (g) insequence No. 2 and adenine (a) in sequence No. 3, and base No. 1242 isadenine (a) in sequence No. 2 and guanine (g) in sequence No. 3.

Therefore, the microorganism of the present invention is characterizedin having the base sequence of sequence No. 1 including theabove-mentioned sequences No. 2 and No. 3 (that is, base No. 444 andbase No. 1242 are represented by “r”) from the 5′ terminal side of 16SrDNA.

In the present invention, the 16S rDNA base sequence was analyzed asbelow.

InstaGene Matrix (produced by BIO RAD Laboratories, Inc., California(CA), U.S.A.) was used for DNA extraction; PrimeSTAR HS DNA Polymerase(produced by Takara Bio Inc.) was used for PCR; BigDye Terminator v3.1Cycle Sequencing Kit (produced by Applied Biosystems, California (CA),U.S.A.) was used to determine cycle sequence, respectively. The usedprimers (in accordance with “Gene Analysis Method—method for determiningthe base sequence of 16S rRNA gene”, Yasuyoshi Nakagawa et al., editedby the Society for Actinomycetes Japan, Classification andidentification of Actinomycetes, pp. 88-117, Business Center forAcademic Societies Japan, 2001) were 9F, 339F, 785F, 1099F, 536R, 802R,1242R and 1541R. The sequence was identified using ABI PRISM 3100Genetic Analyzer System (produced by Applied Biosystems, California(CA), U.S.A.).

As a result of homology search on the basis of the international basesequence database (GenBank/DDBJ/EMBL) using BLAST (ALTSCHUL, (S. F.) etal., Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs. Nucleic Acid Res. 1997.25, 3389-3402), the basesequence of 16S rDNA of AT-332 strain and AT-79 strain had high degreeof homology with the 16S rDNA derived from Bacillus genus, and both ofthe strains had the highest homology of 99.9% with 16S rDNA of Bacillusamyloliquefaciens BCRC11601 strain. On the other hand, as a result ofthe homology search on the basis of the international base sequencedatabase (GenBank/DDBJ/EMBL), no 16S rDNA base sequence of AT-332 andAT-79 strains did exactly match the 16S rDNA base sequence derived fromBacillus genus.

In the present invention, molecular phylogenetic analysis was performedas below.

16S rDNA derived from the standard strain from the strain group whichwas assumed to be closely-related was obtained from the internationalbase sequence database (GenBank/DDBJ/EMBL) to perform molecularphylogenetic analysis using 1500 bp of the 16S rDNA base sequenceobtained in the above.

16S rDNA used for the molecular phylogenetic analysis were derived fromthe following strains.

-   -   Bacillus subtilis, IAM12118T (AB042061)    -   Bacillus subtilis subsp. spizizenii, NBRC101239T (AB325584)    -   Bacillus mojavensis, IF015718 T (AB021191)    -   Bacillus vallismortis, DSM11031 T (AB021198)    -   Bacillus amyloliquefaciens, BCRC11601 T (EF433406)    -   Bacillus atrophaeus, JCM9070 T (AB021181)    -   Bacillus aerophilus, 28K T (AJ831844)    -   Bacillus sonorensis, BCRC17416 T (EF433411)    -   Bacillus licheniformis, DSM13 T (AE017333)    -   Bacillus altitudinis, 41KF2b T (AJ831842)    -   Bacillus cereus ATCC14579 T (NC_004722)BSL2

“T” at the end of the strain name means the standard strain of thespecies. BSL means that the strain is at a bio safety level (level 2 orhigher is indicated). The codes in the brackets indicate the accessionnumber.

The obtained molecular phylogenetic tree is shown in FIG. 1.

The numbers near the branches are the bootstrap values and a scale baris shown at the lower left.

Since AT-332 strain and AT-79 strain have the property that does notcarry out nitrate reduction as mentioned above, their mycologicalcharacteristics did not exactly match those of the Bacillusamyloliquefaciens described in Bergey's Manual. Also, from the result ofthe 16S rDNA analysis, the AT-332 strain and AT-79 strain are consideredto be closely related to Bacillus amyloliquefaciens but cannot beidentified as Bacillus amyloliquefaciens and AT-332 strain and AT-79strain was determined to be a novel strain belonging to Bacillus genus.

The Bacillus sp. AT-332 strain and AT-79 strain of the present inventionare allowed to grow by known means such as the static culture on a solidmedium and the liquid culture and the kind of the available medium,culture conditions and the like are not particularly limited as long asthey allow the bacteria to survive and grow. Examples of the mediuminclude a medium containing glucose, peptone, yeast extract and the likeas well as a general medium such as a meat extract. Also, other than aliquid medium, a solid medium such as an agar slant medium and a platemedium other than a liquid medium may be used.

All the carbon sources which the AT-332 strain and AT-79 strain canutilize can be used for the medium. Specific examples include varioussynthetic or natural carbon sources which the AT-332 strain and AT-79strain can utilize other than sugars such as glucose, galactose,lactose, sucrose, maltose, malt extracts, waste molasses, starch syrupand starch hydrolysate.

Similarly, various synthetic and natural substances which theabove-mentioned strains can utilize such as an organicnitrogen-containing substance including peptone, meat extrat, yeastextract, soy-bean powder and corn steep liquor can be used for thenitrogen source of the medium.

According to a conventional method for culturing microorganisms,inorganic salts such as dietary salt and phosphoric salt, salts of metalsuch as calcium, magnesium and iron and micronutrients such as vitaminsand amino acids can be added as needed.

The culture can be performed under an aerobic condition such as theshake culture and aeration culture. The culture temperature is 20 to 40°C. and preferably 25 to 35° C., pH is 5 to 8 and preferably 6 to 7, andthe culture period is one to four days and preferably two to three days.

The culture containing the bacterial body of the Bacillus sp. AT-332strain and AT-79 strain of the present invention has the property ofcontrolling various plant diseases, controlling nematodes and promotinggrowth of useful plants.

Various plant diseases can be prevented and nematodes can be controlledby allowing the processed product of the culture containing thebacterial body of the Bacillus sp. AT-332 strain and AT-79 strain of thepresent invention, mixture of the culture and other components and thelike; the processed product of separated cultured bacteria cellsobtained by subjecting the culture product to centrifugal separationtreatment or by washing the bacteria cells, the mixture of separatedcultured bacteria cells and other components, and the like; a diluentthereof with a liquid or a solid and the like to exist on the plant bodysuch as roots, stems, leaves, seeds and fruits or in the grove soil.

The Bacillus sp. AT-332 strain and AT-79 strain of the present inventionis available as a plant disease control agent, a nematode control agentand a plant disease promoter in any state of nutritive cells, spores orthe mixture of both as long as the bacteria are living. Also, thestrains can be used if the components of the culture medium are mixed asthey are after the cultivation or if they are in a state where thecomponents other than bacteria cells are removed by washing withdistilled water and the like.

The Bacillus sp. AT-332 strain and AT-79 strain of the present inventioncan control the plant disease caused by fungi and bacteria belonging toOomycetes, Ascomycetes, Basidiomycetes and Deuteromycetes depending onthe type of application and can control phytoparasitic nematode such asDitylenchus dipasaci, Ditylenchus destructor, Pratylenchus sp.,Meloidogyne sp., Heterodera sp. and Globodera spp. The strains canpromote the growth of crops, vegetables, fruits, flowers and legumes atthe same time.

Specifically, the offending bacteria which the Bacillus sp. AT-332strain and AT-79 strain of of the present invention can control includePyricularia oryzae, Cochliobolus miyabeanus, Rhizoctonia solani andGibberella fujikuroi which infest rice; Erysiphe graminis f.sp. hordei,Erysiphe graminis f.sp. tritici, Puccinia striiformis, Pucciniagraminis, Puccinia recondita f.sp. tritici, Puccinia hordei, Gibberellazeae, Pyrenophorateres, Typhula incarnata, Typhula ishikariensis,Sclerotiniaborealis, Micronectriella nivalis, Ustilago nuda, Tilletiacaries, Tilletia toetida, Tapesia yallundea, Phynchosporium secalisf.sp. hordei, Septoria tritici and Lentosphaeria nodorum which infestwheats; Diaporthe citri, Elsinoe fawcettii, Phytophthora citrophthora,Penicillium digitatum and Penicillium italicum of citrus plants;Monilinia mali, Valsa ceratosperma, Podosphaera leucotricha, Alternariaalternata apple pathotype, Venturia inaequalis, Gymnosporangium yamadae,Botriophaeria berengeriana f.sp. piricola, Zygophiala jamaicensis,Gloeodes pomigena, Mycosphaerella pomi, Glomerella cingulata andDiplocarponmali of apples; Venturia nashicola, Alternaria alternatajapanese pear pathotype, Physalospora piricola and Gymnosporangiumasiaticum of pears; Monilinia fructicola, Cladosporium carpophilum andPhomopsis sp. of peaches; Pseudocercospora vitis, Marssonina viticola,Elsinoe ampelina, Glomerella cingulata, Uncinula necator, Phakopsoraampelopsidis and Phomopsis sp. of grapes; Phyllactinia kakicola,Colletotrichum gloeosporioides, Cercospora kaki and Mycosphaerella nawaeof persimmons; Cladosporium carpophilum of plums; Monilinia fructicolaof Prunus avium; Sphaerotheca fuliginea, Didymella bryoniae,Colletotorichum legenarium of gourds; Alternaria solani, Cladosporiumfulvum of tomatoes; Phomopsis vexans and Erysiphe cichoracearum ofeggplants; Alternaria japonica, Alternaria bracicae, Alternariabrassicicola and Cercosporella brassicae of brassica vegetables;Pucciniaallii of green onions; Pyrhium ultimum and Pythium zigiberis ofgingers; Sphaerotheca humuli and Glomerella cingulata of strawberries;Cercospora kikuchii, Elsinoe glycines and Diaporthe phaseolorum var.sojae of soybeans; Cercospora canescens and Uromyces phaseoli var.azukicola of azuki beans; Colletotrichum lindemuthianum of marrow beans;Cercosporidium personatum, Cercospora arachidicola and Shacelomaarachidis of peanuts; Erysiphe pisi of peas; Alternaria solani ofpotatoes; Exobasidium reticulatum, Elsinoe leucospila, Pestalotiopsistheae and Pestalotiopsis longiseta of teas; Alternaria longipes,Erysiphe cichoracearum and Colletotrichum gloeosporioides of tobaccos;Cercospora beticola of sugar beets; Curvularia geniculata andCeratobasidium spp. of the lawn grass; Diplocarpon rosae and Shaerothecapannosa of roses; Septoria obesa and Puccinia horiana of chrysanthemums;and Botrytis cinerea and Sclerotinia sclerotiorum of various cropplants, but not limited thereto.

The plant disease control agent of the present invention includes apostharvest disease control agent for the stored crops after harvesting,particularly in order to prevent fruits and the like from decay. Thereis no limit on the kinds of crops to which the postharvest diseasecontrol agent of the present can be applied, and examples include fruitssuch as strawberry, grape, fig, citrus, peach, melon, watermelon, apple,pear, banana and pineapple and vegetables such as a cucumber, tomato,Chinese cabbage, cabbage, Welsh onion, onion, carrot, Japanese radish,ginger, green pepper, eggplant, pumpkin and bean sprout. There is nolimit on the kinds of fungi which cause the postharvest disease andexamples include Botrytis cinerea, Colletotrichum gloeosporioides andAlternaria alternata.

Examples of nematodes which the Bacillus sp. AT-332 strain and AT-79strain of the present invention can control include Meloidogyne sp. suchas Meloidogyne hapla, Meloidogyne incognita, Meloidogyne javanica andMeloidogyne species; Globodera spp. such as Globodera rostochiensis andother Globodera species; Heterodera sp. such as Heterodera avenae,Heterodera glycines, Heterodera schachtii, Heterodera trifolii and otherHeterodera species; Anguiana species belonging to Anguina funesta;Aphelenchoides species; Belonolaimus longicaudatus and other speciesbelonging to Belonolaimus; Bursaphelenchus xylophilus belonging toBursaphelenchus xylophilus and other Bursaphelenchus species; Criconemaspecies, Criconemella species, Criconemoides species and Mesocriconemaspecies belonging to Criconemoides; Ditylenchus destructor, Ditylenchusdipsaci and other species belonging to Ditylenchus; Dolichodorus speciesbelonging to awl nematodes; Heliocotylenchus multicinctus belonging toHelicotylenchus and other Helicotylenchus species; Hemicycliophoraspecies and Hemicriconemoides species belonging to sheath and sheathoidnematodes; Hirshmanniella species; Hoploaimus species belonging toHoplolaimus; Nacobbus species belonging to Nacobbus; Longidoruselongatus belonging to Longidorus and other Longidorus species;Pratylenchus neglectus, Pratylenchus penetrans, Pratylenchus curvitatus,Pratylenchus goodeyi and other Pratylenchus species belonging toPratylenchus sp.; Radopholus similis and other species belonging toRadopholus; Rotylenchus robustus and other Rotylenchus species belongingto Rotylenchulus reniformis; Scutellonema species; Trichodorusprimitivus and other Trichodorus species belonging to stubby rootnematodes; Paratrichodorus species; Tylenchorhynchus claytoni,Tylenchorhynchus dubius and other species belonging to Tylenchorhynchus;Tylenchulus species belonging to Tylenchulus semipenetrans; andXiphinema species belonging to Xiphinema americanum, but not limitedthereto.

The Bacillus sp. AT-332 strain and AT-79 strain of the present inventionare especially useful for controlling Meloidogyne species, Globoderaspecies, Heterodera species, Pratylenchus species, Radopholus species,Rotylenchus species and Tylenchulus species, and in particulay can besuitably used for exterminating Meloidogyne species, Pratylenchusspecies, Globodera species and Heterodera species.

Examples of crops which the Bacillus sp. AT-332 strain and AT-79 strainof the present invention can promote growth include cereal crops such asrice, wheat and corn; vegetables such as carrot, cucumber, Japaneseradish, pumpkin, lettuce, eggplant, tomato, cabbage, potato, Chinesecabbage, crown daisy, Japanese mustard spinach, green pepper, Welshonion, onion, ginger, garlic, strawberry; mushrooms such as shiitakemushroom; fruit trees such as persimmon, pear, orange, grape, apple andpeach; flowers and ornamental plants such as chrythansemum, tulip androse; and legumes such as soybean, sesame and peanut.

The plant disease control agent, nematode control agent and plant growthpromoter of the present invention contains the Bacillus sp. AT-332strain and AT-79 strain which can control the plant diseases andnematodes and have an effect of promoting plant growth as mentionedabove as an indicated microorganism.

In the plant disease control agent, nematode control agent and plantgrowth promoter of the present invention, the AT-332 strain or AT-79strain can be used singly or in combination. Also, the mutant of each ofthe strains can be used. The mutant is the one that possesses theabove-mentioned bacteriological property of the AT-332 strain and AT-79and an activity of controlling the plant diseases, controlling nematodesand promoting plant growth. A natural mutant strain, a mutant straincaused by ultraviolet ray or a chemical mutagenesis agent, a cell fusionstrain and a genetically-modified strain or the like can be used.

When the live bacteria of the AT-332 strain and AT-79 strain are used inthe plant disease control agent, nematode control agent and plant growthpromoter of the present invention, it is preferable to add the bacteriato the plant body at a concentration of 10⁵ to 10¹⁰ units/ml.

When the culture product of the AT-332 strain and/or AT-79 strain isused, the dosage can be appropriately determined in individual cases ofthe above-mentioned viable bacteria.

As the microbiological agent (plant disease control agent, nematodecontrol agent and plant growth promoter) of the present invention, thebacteria cells and/or culture product of the AT-332 strain and AT-79strain can be used singly. Or the microbiological agent can be dilutedwith an inert liquid or a solid carrier to be used as a pharmacologicalagent with addition of the surfactant, dispersing agent and otheradjuvant as needed. Examples of specific formulation include granularformulation, dust formulation, wettable powder, suspension agent andemulsion formulation.

Examples of the carrier include talc, bentonite, kaolin, clay, diatomearth, white carbon, vermiculite, lime hydrate, ammonium sulfate, silicasand, urea, a porous solid carrier and liquid carriers such as water,isopropyl alcohol, methyl naphthalene, xylene, cyclohexanone andalkylene glycol. Examples of the surfactant and dispersion agent includedinaphthylmethanesulfonic acid salts, alcohol sulfuric acid ester salts,lignin sulfonic acid salts, alkylarylsulfonic acid salts,polyoxyethylene glycol ethers, polyoxyethylene sorbitan monoalkylate andpolyoxyethylene alkylaryl ethers. Examples of the adjuvant includecarboxymethylcellulose, polyethylene glycol, propylene glycol, gumArabic and xanthan gum; and examples of the cryoprotective agent includeskim milk and pH buffering agent. The amount of the live bacteria and/orculture product of the AT-332 strain and AT-89 strain, the time ofapplication and the application amount can be appropriately determineddepending on each case of the above viable bacteria.

The microbiological agent (plant disease control agent, nematode controlagent and plant growth promoter) of the present invention can containactive ingredients other than those of the present invention: i.e.insecticides, other bactericidal agents, herbicides, plant growthregulators and fertilizers. Also, the plant disease control agent,nematode control agent and plant growth promoter of the presentinvention may contain the strain of other species in combination withthe AT-332 strain and/or AT-79 strain.

Examples of the bactericidal components include bitertanol,bromuconazole, cyproconazole, difenoconazole, diniconazole,enilconazole, epoxiconazole, fluquinconazole, fenbuconazole,flusilazole, flutriafol, hexaconazole, imibenconazole, ipconazole,metconazole, myclobutanil, penconazole, propiconazole, prothioconazole,simeconazole, triadimefon, triadimenol, tebuconazole, tetraconazole,triticonazole, prochloraz, pefurazoate, imazalil, triflumizole,cyazofamid, benomyl, carbendazim, thiabendazole, fuberidazole,ethaboxam, etridiazole, oxypoconazole fumaric acid, himexazole,azoxystrobin, dimoxystrobin, enestroburin, fluoxastrobin,kresoxym-methyl, metominostrobin, oryzastrobin, picoxystrobin,pyraclostrobin, trifloxystrobin, carboxin, benalaxyl, boscalid, bixafen,fenhexamid, flutolanil, furametpyr, mepronil, metalaxyl, mefenoxam,ofurace, oxadixyl, oxycarboxin, penthiopyrad, thifluzamide, tianidil,dimethomorph, flumorph, flumetover, fluopicolide, carpropamid,diclocymet, mandipropamid, fluazinam, pyrifenox, bupirimate, cyprodinil,fenarimol, ferimzone, mepanipyrim, nuarimol, pyrimethanil, triforine,fenpiclonil, fludioxonil, aldimorph, dodemorph, fenpropimorph,tridemorph, fenpropidin, iprodione, procymidone, vinclozolin,famoxadone, fenamidone, octhilinone, probenazole, anilazine,diclomezine, pyroquilon, proquinazid, tricyclazole, captafol, captan,dazomet, folpet, fenoxanil, quinoxyfen, amisulbrom, manzeb, maneb,metam, metiram, ferbam, propineb, thiuram, zineb, ziram, diethofencarb,iprovalicarb, benthiavalicarb-isopropyl, propamocarb hydrochloride,thiophanate methyl, pyribencarb, Bordeaux mixture, basic copperchloride, basic copper sulfide, cupric hydroxide, copper8-hydroxyquinoline, dodine, iminoctadine albesilate, iminoctadineacetate, guazatine, kasugamycin, streptomycin, polyoxin,oxytetracycline, validamycin A, binapacryl, dinocap, dinobuton,dithianon, isoprothiolane, edifenphos, iprobenfos, fosetyl, fosetylaluminum, pyrasophos, tolclofos-methyl, chlorothalonil, dichlofluanid,flusulfamide, hexyachlorobenzene, phthalide, pencycuron, quintozene,cyflufenamid, cymoxanil, dimethirimol, ethyrimol, furalaxyl,metrafenone, spiroxamine, amobam, sulfur, lime sulfur, echlomezole,potassium bicarbonate, calcium bicarbonate, thiadiazine, tecloftalam,triazine, copper nonylphenol sulfonate, hydroxy isoxazole, fluoroimide,polycarbamate, methasulfocarb, EDDP, IBP, tolfenpyrad, fluopyram,isotianil and isopyrazam, but not limited thereto.

Examples of the insecticidal components include acetamiprid,pymetrozine, fenitrothion, acephate, carbaryl, methomyl, cartap,cyhalothrin, ethofenprox, teflubenzuron, flubendiamide, flufenoxuron,tebufenozide, fenpyroximate, pyridaben, imidacloprid, buprofezin, BPMC,MIPC, malathion, methidathion, fenthion, daiazinon, oxydeprofos,vamidothion, ethiofencarb, pirimicarb, permethrin, cypermethrin,bifenthrin, halfenprox, silafluofen, nitenpyram, chlorfluazuron,methoxyfenozide, tebufenpyrad, pyrimidifen, kelthane, propargite,hexythiazox, clofentezine, spinosad, milbemectin, BT (Bacillusthuringiensis), indoxacarb, metaflumizone, chlorfenapyr, fipronil,etoxazole, acequinocyl, pirimiphos-methyl, acrinathrin, quinomethionate,chlorpyrifos, abamectin, emamectin benzoate, fenbutatin oxide, terbufos,ethoprophos, cadusafos, fenamiphos, fensulfothion, DSP, dichlofenthion,fosthiazate, oxamyl, isoamidofos, fosthietan, isazophos, thionazin,benfuracarb, spirodiclofen, ethiofencarb, azinphos-methyl, disulfoton,methiocarb, oxidemethon-methyl, parathion, cyfluthrin, beta-cyfluthrin,tebupirimfos, spiromesifen, endosulfan, amitraz, tralomethrin,acetoprole, ethiprole, ethion, triclorfon, methamidophos, dichlorvos,mevinphos, monocrotophos, dimethoate, formetanate, formothion, mecarbam,thiometon, disulfoton, naled, methyl parathion, cyanophos, diamidafos,albendazole, oxibendazole, fenbendazole, oxfendazole, propaphos,sulprofos, prothiofos, profenofos, isofenphos, temephos, phenthoate,dimethylvinphos, chlorfenvinphos, tetrachlorvinphos, phoxim, isoxathion,pyraclofos, chlorpyrifos, pyridaphenthion, phosalone, phosmet,dioxabenzofos, quinalphos, pyrethrin, allethrin, prallethrin,resmethrin, permethrin, tefluthrin, fenpropathrin, alpha-cypermethrin,lambda-cyhalothrin, delta-methrin, fenvalerate, esfenvalerate,flucythrinate, fluvalinate, cycloprothrin, thiodicarb, aldicarb,alanycarb, metolcarb, xylylcarb, propoxur, fenoxycarb, fenothiocarb,bifenazate, carbofuran, carbosulfan, sulfur, pyrifluquinazon,furathiocarb, diafenthiuron, diflubenzuron, hexaflumuron, novaluron,lufenuron, chlorfluazuron, tricyclohexyltin hydroxide, sodium oleate,potassium oleate, methoprene, hydroprene, binapacryl, amitraz,chlorobenzilate, phenisobromolate, tetradifon, bensultap, benzomate,chromafenozide, halofenozide, endosulfan, diofenolan, tolfenpyrad,triazamate, nicotine sulfate, thiacloprid, thiamethoxam, clothianidin,dinotefuran, fluazinam, pyriproxyfen, fluacrypyrim, hydramethylnon,cyromazine, TPIC, thiocyclam, fenazaquin, polynactin complex,azadirachtin, rotenone, hydroxypropyl starch, mesulfenphos, phosphocarb,isoamidofos, aldoxycarb, metham sodium, morantel tartrate, dazomet,levamisole hydrochloride, trichlamide, tolfenpyrad, pyridalyl,chlorantraniliprole, cyenopyrafen and cyflumetofen, but not limitedthereto.

The plant disease control agent, nematode control agent and plant growthpromoter of the present invention can be directly applied as they are orapplied as a solution diluted with water and the like. The applicationmethod of the plant disease control agent, nematode control agent andplant growth promoter of the present invention is not particularlylimited and examples thereof include a method of spraying the agentdirectly on plants and insect pests, a method of spraying the agent onthe soil, a method of adding the agent to the water and fertilizer to beapplied on plants and the soil and a method of coating the seeds withthe agent. In addition, it is desirable to appropriately adjust theapplication amount of the drug product since the application amountvaries depending on the disease and the insect pest to be controlled,the crops as the subject of the application, the application method,occurrence tendency of diseases, degree of the damage, environmentalconditions and the formulations to be used.

As discussed above, the AT-332 strain and AT-79 strain of the presentinvention have a broad disease and nematicidal spectrum and can controlvarious kinds of plant diseases and nematodes, and can promote plantgrowth. Since the plant disease control agent, nematode control agentand plant growth promoter of the present invention comprising thesestrains are highly safe for the environment and has control effects onvarious kinds of diseases and nematodes, the plant disease control agentcan prevent a wide range of diseases and nematodes without using othermeans in combination and can be used as a biological pesticide and/orbiological fertilizer which can promote the growth of useful plants aswell.

EXAMPLES

The present invention is to be described in more details with ProductionExample, Formulation Examples, Examples and Comparative Examples, butthe present invention is not limited to these examples.

Culture of AT-332 Strain and AT-79 Strain

The AT-332 strain and AT-79 strain were isolated from the soilcontaining plant roots.

In detail, 1 g of a dry soil obtained by collecting the soil in MoriyaCity in Ibaraki Prefecture, Japan in August 2009 and subjecting it toheat treatment (80° C., for 10 minutes) was suspended in the sterilizedwater. The suspension was diluted with the dilution rate of 10² to 10⁴times and the separate culture of the suspension was carried out on thenutrient broth medium (Eiken Chemical Co., Ltd.) (28° C., for threedays) and the formed colonies were isolated. The isolated colonies werecultured on a potato dextrose agar medium and the strains effectiveagainst pathogens of various plant diseases were found. The strains werefurther subjected to shaking culture on a potato dextrose liquid medium,and the Bacillus sp. AT-332 strain and AT-79 strain were isolated as astrain having an activity against the second-stage larva of sweetpotatoMeloidogyne sp.

The method for identification of each of the strains, the variousanalysis methods and results thereof, and bacteriological properties arethose as described in “Mode for Carrying out the Invention”.

Production Example 1 Cultivation and Preparation of AT-332 Strain

As a preculture, one loopful of the preserved bacteria of the presentinvention (AT-332 strain) was inoculated on 60 ml per flask of anutrient broth medium (available from Eiken Chemical Co., Ltd.) in a 500ml conical flask with baffles, and subjected to shaking culture using arotary shaker at 180 rpm and 28° C. for one day.

60 ml of the culture obtained by the above preculture was inoculated ina jar fermentor with a 5000 ml volume containing a 2,000 ml of LB medium(20 g of peptone, 10 g of yeast extract, 20 g of sodium chloride andwater for the rest) and cultivated as the main culture at 500 rpm,aeration rate of 1 l/hour and 35° C. for three days.

About 1,800 g of culture was obtained by the above main culture. Theconcentration of the bacteria cell was about 8.0×10⁹ CFU/ml.

About 140 g of dry powder was obtained by freezing 1,800 g of theobtained culture product at −80° C., followed by freeze-drying underreduced pressure and pulverization. The bacteria cell concentration ofthe powder was about 1.0×10¹¹ CFU/g.

Production Example 2 Cultivation and Preparation of AT-79 Strain

As a preculture, one loopful of the preserved bacteria of the presentinvention (AT-79 strain) was inoculated on 60 ml per flask of a nutrientbroth medium (available from Eiken Chemical Co., Ltd.) in a 500 mlconical flask with baffles, and subjected to shaking culture using arotary shaker at 180 rpm and 28° C. for one day.

60 ml of the culture obtained by the above preculture was inoculated ina jar fermentor with a 5000 ml volume containing a 2,000 ml of LB medium(20 g of tryptone, 10 g of yeast extract, 20 g of sodium chloride andwater for the rest) and cultivated as the main culture at 500 rpm,aeration rate of 1 l/hour and 35° C. for three days.

About 1,700 g of culture was obtained by the above main culture. Thebacteria cell concentration of the powder was about 9.0×10⁹ CFU/g.

About 130 g of dry powder was obtained by freezing 1,700 g of theobtained culture product at −80° C., followed by freeze-drying underreduced pressure and pulverization. The bacteria cell concentration ofthe powder was about 1.0×10¹¹ CFU/g.

Formulation Examples are given below. Here, the word “part(s)” means apart(s) by mass.

Formulation Example 1 Wettable Powder

60 parts of dry powder obtained by Formulation Example 1, 25 parts ofdiatom earth, 5 parts of white carbon, 8 parts of lignin sulfonate and 2parts of alkyl naphthalene sulfonate were mixed and pulverized tothereby obtain wettable powder.

Formulation Example 2 Granular Formulation

5 parts of dry powder obtained by Formulation Example 1, 25 parts ofbentonite, 66 parts of talc, 2 parts of dodecylbenzene sulfonate and 2parts of lignin sulfonate were mixed and pulverized. After adding about20 parts of water thereto and kneading the mixture by a kneadingmachine, the resultant was granulated by a granulator and dried, andthen the size of the granules was regulated to obtain a granularformulation.

Formulation Example 3 Wettable Powder

60 parts of dry powder obtained by Formulation Example 2, 25 parts ofdiatom earth, 5 parts of white carbon, 8 parts of lignin sulfonate and 2parts of alkyl naphthalene sulfonate were mixed and pulverized tothereby obtain wettable powder.

Formulation Example 4 Granular Formulation

5 parts of dry powder obtained by Formulation Example 2, 25 parts ofbentonite, 66 parts of talc, 2 parts of dodecylbenzene sulfonate and 2parts of lignin sulfonate were mixed and pulverized. After adding about20 parts of water thereto and kneading the mixture by a kneadingmachine, the resultant was granulated by a granulator and dried, andthen the size of the granules was regulated to obtain a granularformulation.

Next, Examples and Comparative Examples for testing the effects of theplant disease control agent, nematode control agent and plant growthpromoter of the present invention are described below.

Example 1 and Comparative Example 1 Test for the Effects Against RiceBlast

Sufficient doses of the diluted wettable powders in Formulation Examples1 and 3 with the dilution rate of 250 times was sprayed with a spray gunon the rice (variety: Koshihikari, 15 plants per hill) grown in aglasshouse to the third-leaf unfolding stage in a plastic pot 6 cm indiameter. As a comparative example, the Impression wettable powder(produced by SDS Biotech K.K.) with the dilution rate of 250 times wasalso subjected to the test in the same manner. The next day, suspensionof the rice blast pathogen (Pyricularia oryzae) spores was sprayed andinoculated. After retaining the pots in a humidity room at 22° C. for 24hours, the pots were allowed to stand in greenhouse for seven days andthe number of lesions in the inoculated leaves was investigated tothereby determine the control titer. The control titer (%) wascalculated on the basis of the number of lesions of the leaves in thenon-treated region. As can be seen from the results shown in Table 1, bythe treatment with the microbiological agent of the present invention,the incidence of rice blast was greatly reduced compared to thenon-treated region, and significantly high control effects wereobtained.

TABLE 1 Number of Control titer lesions (%) Treated region (FormulationExample 1) 28 86.0 Treated region (Formulation Example 3) 20 90.0Comparative region (Impression wettable 85 57.5 powder) Non-treatmentregion 200 0.0

Example 2 and Comparative Example 2 Test for the Effects on CucumberAnthracnose

Sufficient doses of the wettable powders in Formulation Examples 1 and 3with the dilution rate of 250 times were sprayed by a spray gun on thefirst and second leaves of cucumbers (variety: Tokiwa Hikari No. 3p-type) grown in a glasshouse to the third-leaf unfolding stage in aplastic pot 6 cm in diameter. As a comparative example, the diluent ofImpression wettable powder (produced by SDS Biotech K.K.) with thedilution rate of 250 times was also subjected to the test in the samemanner. The next day, suspension of the cucumber Colletorichumlagenarium spores was sprayed and inoculated. After retaining the potsin a humidity room at 22° C. for 24 hours, the pots were allowed tostand in greenhouse for seven days and the diseased area rate in thefirst and second leaves was investigated with eyes to thereby determinethe control titer. The control titer (%) was calculated on the basis ofthe diseased area rate in the non-treatment region. As can be seen fromthe results in Table 2, by the treatment with the microbiological agentof the present invention, the incidence of cucumber Colletorichumlagenarium was greatly reduced compared to the non-treated region, andsignificantly high control effects were obtained.

TABLE 2 Diseased Control titer area rate (%) Treated region (FormulationExample 1) 7 82.5 Treated region (Formulation Example 3) 8 80.0Comparative region (Impression wettable 25 37.5 powder) Non-treatmentregion 40 0.0

Example 3 and Comparative Example 3 Test for the Effects on TomatoPhytophthora infestans

Sufficient doses of the wettable powders in Formulation Examples 1 and 3with the dilution rate of 250 times were sprayed by a spray gun on thetomatoes (variety: Sugar lump) grown in a glasshouse to the fifth-leafunfolding stage in a plastic pot 6 cm in diameter. As a comparativeexample, the diluent of Impression wettable powder (produced by SDSBiotech K.K.) with the dilution rate of 250 times was also subjected tothe test in the same manner. The next day, suspension of the tomatoPhytophthora infestans zoospores was sprayed and inoculated. Afterretaining the pots in a humidity room at 22° C. for 16 hours, the potswere allowed to stand in greenhouse for three days and the diseased arearate in the third, fourth and fifth leaves was investigated with eyes tothereby determine the control titer. The control titer (%) wascalculated on the basis of the diseased area rate in the non-treatmentregion. As can be seen from the results in Table 3, by the treatmentwith the microbiological agent of the present invention, the incidenceof tomato Phytophthora infestans was greatly reduced compared to thenon-treated region, and significantly high control effects wereobtained.

TABLE 3 Diseased Control titer area rate (%) Treated region (FormulationExample 1) 7 84.4 Treated region (Formulation Example 3) 6 86.7Comparative region (Impression wettable 40 11.1 powder) Non-treatmentregion 45 0.0

Example 4 and Comparative Example 4 Test for the Effects on CucumberPseudoperonospora cubensis

Sufficient doses of the wettable powders in Formulation Examples 1 and 3with the dilution rate of 250 times were sprayed by a spray gun on thecucumbers (variety: Hikari No. 3 p-type) grown in a glasshouse to thethird-leaf unfolding stage in a plastic pot 6 cm in diameter. As acomparative example, the diluent of Impression wettable powder (producedby SDS Biotech K.K.) with the dilution rate of 250 times was alsosubjected to the test in the same manner. The next day, suspension ofthe cucumber Pseudoperonospora cubensis zoospores was sprayed andinoculated. After retaining the pots in a humidity room at 22° C. for 18hours, the pots were allowed to stand in greenhouse for three days andthe diseased area rate in the first and second leaves was investigatedwith eyes to thereby determine the control titer. The control titer (%)was calculated on the basis of the diseased area rate in thenon-treatment region. As can be seen from the results in Table 4, by thetreatment with the microbiological agent of the present invention, theincidence of cucumber Pseudoperonospora cubensis was greatly reducedcompared to the non-treated region, and significantly high controleffects were obtained.

TABLE 4 Diseased Control titer area rate (%) Treated region (FormulationExample 1) 4 88.6 Treated region (Formulation Example 3) 3 91.4Comparative region (Impression wettable 25 28.6 powder) Non-treatmentregion 35 0.0

Example 5 and Comparative Example 5 Test for the Effects on AppleAltenaria Alternaria Mali

Leaves of apples (variety: Orin) were collected and sufficient doses ofthe wettable powders in Formulation Examples 1 and 3 with the dilutionrate of 250 times were sprayed by a spray gun on the back side of theleaves. As a comparative example, the diluent of Impression wettablepowder (produced by SDS Biotech K.K.) with the dilution rate of 250times was also subjected to the test in the same manner. After spraying,the leaves were air-dried and the suspension of the apple AltenariaAlternaria mali spores was sprayed and inoculated thereto. After theleaves were left to stand at 20° C. in humid condition for four days,the diseased area rate was investigated with eyes to thereby determinethe control titer. The control titer (%) was calculated on the basis ofthe diseased area rate in the non-treatment region. As can be seen fromthe results in Table 5, by the treatment with the microbiological agentof the present invention, the incidence of apple Altenaria Alternariamali was greatly reduced compared to the non-treated region, andsignificantly high control effects were obtained.

TABLE 5 Diseased Control titer area rate (%) Treated region (FormulationExample 1) 5 91.7 Treated region (Formulation Example 3) 7 88.3Comparative region (Impression wettable 30 50.0 powder) Non-treatmentregion 60 0.0

Example 6 and Comparative Example 6 Test for the Effects on CucumberSphaerotherca fuliginea (Field Test

The test was performed in the company-owned greenhouse using cucumbers(test region: 4 m²/region; 10 plants/region; in triplicate). The diseasewas allowed to occur naturally. The wettable powders in FormulationExamples 1 and 3 with the dilution rate of 500 times, 1,000 times and2,000 times were sprayed four times at intervals of seven days and thecontrol titer (%) was calculated from the disease area rate on theleaves. The Impression wettable powder (SDS Biotech K.K.) with thedilution rate of 500 times and 1,000 times, Botokiller wettable powder(Idemitsu Kosan Co., Ltd.) with the dilution rate of 1,000 times,Botopika wettable powder (Idemitsu Kosan Co., Ltd.) with the dilutionrate of 2,000 times, Ecoshot granule wettable powder (Kumiai ChemicalIndustry Co., Ltd.) with the dilution rate of 1,000 times and Morestanwettable powder (Agro-Kanesho Co., Ltd.) with the dilution rate of 3,000times were used as a comparative agent. The incidence of the disease inthe non-treated region was 47.4%. The control titer (%) was calculatedon the basis of the incidence in the non-treatment region. As can beseen from the results in Table 6, by the treatment with themicrobiological agent of the present invention, the incidence ofcucumber Sphaerotherca fuliginea was greatly reduced compared to thenon-treated region, and significantly high control effects wereobtained. A remarkably higher effect was confirmed in the field as wellcompared to conventional commercially-available Bacillus subtilis agents(Impression wettable powder (Patent Document 3), Botokiller wettablepowder, Botopica wettable powder, Ecoshot wettable powder (PatentDocument 4)) used as a comparative agent. The microbiological agent ofthe present invention with the dilution rate of 500 times showed a veryhigh effect equivalent to the Morestan wettable powder, which is achemical agent.

TABLE 6 Control Incidence titer Treated region (Formulation Example 1),dilution 2.1 95.6 rate: 500 times Treated region (Formulation Example1), dilution 6.2 86.9 rate: 1,000 times Treated region (FormulationExample 1), diluent 10.3 78.3 rate: 2,000 times Treated region(Formulation Example 3), dilution 4.2 91.1 rate: 500 times Treatedregion (Formulation Example 3), dilution 5.7 88.0 rate: 1,000 timesTreated region (Formulation Example 3), dilution 10 78.9 rate: 2,000times Comparative Region (Impression wettable powder), 17.9 62.2dilution rate: 500 times Comparative Region (Botokiller wettablepowder), 35.3 25.5 dilution rate: 1,000 times Comparative Region(Botopika wettable powder), 34.4 27.4 dilution rate: 2,000 timesComparative region (Ecoshot granular wettable 37.5 20.9 powder), diluentrate: 1,000 times Comparative region (Morestan wettable powder) 1.1 97.7diluent rate: 3,000 times Non-treatment region 47.4 0.0

Example 7 and Comparative Example 7 Test for the Effects on EggplantBotrytis cinerea (Field Test)

The test was performed in the company-owned greenhouse using eggplants(test region: 5.6 m²/region; 7 plants/region; in triplicate). Thedisease was allowed to occur naturally. The wettable powders inFormulation Examples 1 and 3 with the dilution rate of 500 times and1,000 times were sprayed four times at intervals of seven days and thecontrol titer (%) was calculated from the incidence in the fruits. TheImpression wettable powder (SDS Biotech K.K.) with the dilution rate of500 times and 1,000 times, Botokiller wettable powder (Idemitsu KosanCo., Ltd.) with the dilution rate of 1,000 times, Botopika wettablepowder (Idemitsu Kosan Co., Ltd.) with the dilution rate of 2,000 times,Ecoshot granule wettable powder (Kumiai Chemical Industry Co., Ltd.)with the dilution rate of 1,000 times and Savior Flowable 20 (SyngentaJapan K.K.) with the dilution rate of 1,500 times were used as acomparative agent. The incidence of the disease in the non-treatedregion was 15%. The control titer (%) was calculated on the basis of theincidence in the non-treatment region. As can be seen from the resultsin Table 7, by the treatment with the microbiological agent of thepresent invention, the incidence of Botrytis cinerea was greatly reducedcompared to the non-treated region, and significantly high controleffects were obtained. A remarkably higher effect was confirmed in thefield as well compared to conventional commercially-available Bacillussubtilis agents (Impression wettable powder, Botokiller wettable powder,Botopica wettable powder, Ecoshot wettable powder) used as a comparativeagent. The microbiological agent of the present invention with thedilution rate of 500 times showed a very high effect equivalent toSavior Flowable 20, which is a chemical agent.

TABLE 7 Control Incidence titer Treated region (Formulation Example 1),dilution 2.4 84.0 rate: 500 times Treated region (Formulation Example1), dilution 4.1 72.7 rate: 1,000 times Treated region (FormulationExample 3), dilution 2.9 80.7 rate: 500 times Treated region(Formulation Example 3), dilution 4.1 72.7 rate: 1,000 times ComparativeRegion (Impression wettable powder), 6.8 54.7 dilution rate: 500 timesComparative Region (Botokiller wettable powder), 8.8 41.3 dilution rate:1,000 times Comparative Region (Botopika wettable powder), 6.9 54.0dilution rate: 2,000 times Comparative region (Ecoshot granular wettable7.2 52.0 powder), diluent rate: 1,000 times Comparative region (SaviorFlowable 20), diluent 2.1 86.0 rate: 1,500 times Non-treatment region 150.0

Example 8 and Comparative Example 8 Test for the Effects on Burkholderiaplantarii

The seed rice (variety: Koshihikari) was immersed to be inoculated inthe suspension of Burkholderia plantarii (1×10⁸ CFU/ml), which wasobtained by the shake culture on the PD liquid medium at 27° C. for 52hours, for one hour under reduced pressure to thereby prepare the seedsinfected with Burkholderia plantarii. The seeds infected withBurkholderia plantarii were immersed in the solution of the wettablepowder of Formulation Example 1 and Formulation Example 3 with thedilution rate of 100 times. After the solution was removed, the seedswere retained in a humidity room of 32° C. for one day to stimulate thegermination. As a comparative agent, the solution of Impression (SDSBiotech K.K.) with the dilution rate of 100 times was also subjected tothe test in the same manner. The germination-stimulated seeds wereseeded in a plastic cup having a diameter of 6 cm filled with culturesoil. The seedlings were retained in a room for raising seedlings at 30°C. for three days after the seeding and in a humidity room at 25° C. for15 days. Then all of the seedlings were investigated for the presence ofthe disease to determine the diseased seedling rate. The control titer(%) was calculated on the basis of the diseased seedling rate in thenon-treated region. The seeding amount per cup was 3 g of dry seed rice(90 to 110 grains). As can be seen from the results in Table 8, by thetreatment with the microbiological agent of the present invention, therate of diseased seedlings of Burkholderia plantarii was greatly reducedcompared to the non-treated region, and significantly high controleffects were obtained.

TABLE 8 Diseased Control titer seedling rate (%) Treated region(Formulation Example 1) 30 60.0 Treated region (Formulation Example 3)35 53.3 Comparative region (Impression wettable 55 26.7 powder)Non-treatment region 75 0.0

Example 9 and Comparative Example 9 Test for the Effect on Rhizoctoniasolani

3 g of the culture product of Rhizoctonia solani in a bran medium wasmixed into 500 ml of sterilized soil to be filled in a plastic pot, and1 g of the granular formulation of Formulation Example 2 and FormulationExample 4 was mixed into the soil, respectively. As a comparative agent,84 mg of Impression wettable powder was also subjected to the test inthe same manner. Cucumbers (variety: Sagami-hanjiro) were seeded andafter growing the cucumbers at 23° C. for one week, the germination ratewas investigated. The control effect (control titer %) was calculated onthe basis of the diseased seedling rate in the non-treated region. Ascan be seen from the results in Table 9, by the treatment with themicrobiological agent of the present invention, the rate of the diseasedseedlings of Rhizoctonia solani was greatly reduced compared to thenon-treated region, and significantly high control effects wereobtained.

TABLE 9 Diseased Control titer seedling rate (%) Treated region(Formulation Example 2) 12 60.0 Treated region (Formulation Example 4)18 40.0 Comparative region (Impression wettable 23 23.3 powder)Non-treatment region 30 0.0

Example 10 and Comparative Example 10 Activity Against the Second-StageLarva of Sweet Potato Meloidogyne sp.

The nematicidal activity against the second-stage larva of sweetpotatoMeloidogyne sp. hatched within 24 hours from the egg capsule collectedfrom the roots of eggplants (variety: Juryo). Each of the solutions ofFormulation 1 and Formulation 3 with the dilution rate of 100 times (aTween 20 solution with the dilution rate of 5,000 times) and anequivalent amount of the second-stage larva of sweetpotato Meloidogynesp. (about 50 worms) were added to a 24-hole microplate. As acomparative agent, the Impression (SDS Bioteck K.K.) solution with thedilution rate of 100 times was also subjected to the test in the samemanner. The plate was sealed and placed in an incubator at 28° C. andrelative humidity of about 50%. After 72 hours, the death rate wasinvestigated by an observation by a stereoscopic microscope. At thattime, immobile nematodes were regarded as being dead. The nematicidalrate was calculated according to the expression described below. As canbe seen from the results in Table 10, by the treatment with themicrobiological agent of the present invention, an extremely highnematicidal activity was obtained against the second-stage larva ofsweetpotato Meloidogyne sp.

Nematicidal rate=(number of dead nematodes/number of testednematodes)×100  [Expression 1]

TABLE 10 Nematicidal rate Treated region (Formulation Example 1) 100Treated region (Formulation Example 3) 100 Comparative region(Impression wettable powder) 10 Non-treatment region 5

Example 11 and Comparative Example 11 Test for the Control EffectAgainst Sweetpotato Meloidogyne Sp.

In a 1/10,000 a-Wagner pot, each of the granular formulation ofFormulation Example 2 and Formulation Example 4 was uniformly mixed inthe soil infected with sweetpotato Meloidogyne sp. at the rate of 40kg/10 a and small-size tomatoes (variety: Sugar lump) were plantedthereto. As a comparative agent, Impression wettable powder (SDS BiotechK.K.) was also subjected to the test in the same manner at the rate of3.3 kg/10 a. One month after the settled planting, the degree of damageto the roots (root-knot degree) was classified and evaluated accordingto the criteria described below. The root-knot index was determinedaccording to the expression as below to calculate the control titer. Ascan be seen from the results in Table 11, by the treatment with themicrobiological agent of the present invention, the root damages causedby sweetpotato Meloidogyne sp. were greatly reduced compared to thenon-treated region, and significantly high control effects wereobtained.

Degree of damage 0: No root-knot was observed.

1: The root-knots are hardly-noticeable at a glance but a few can befound.

2: A few of root-knots are observed.

3: Moderate amount of root-knots are observed.

4: A number of root-knows are observed all over the rhizosphere.

Root-knot index=(Σ(degree of damage×number of units)/all of theinvestigated population×4)×100

Control titer=(1−Root-knot index in the treated region/Root-knot indexin the non-treated region)×100  [Expression 2]

TABLE 11 Degree of Root-knot Control damage index titer Treated region(Formulation Example 2) 1.2 30 70.0 Treated region (Formulation Example4) 2 50 50.0 Comparative region 3.5 87.5 12.5 (Impression wettablepowder) Non-treatment region 4 100 0.0

Example 12 and Comparative Examples 12 to 13 Effect of Promoting PlantGrowth of the AT-332 Strain (Basic Test)

A petri plate basic test was carried out with respect to Arabidopsisthaliana to measure the effect of promoting plant growth of the AT-332strain. After immersing the seeds of Arabidopsis thaliana in 1% sodiumhypochlorite for 20 minutes, the seeds are immersed in 70% ethanolsolution for two minutes to sterilize the surface of the seeds. Afterthat, the seeds were washed with sterile distilled water to be used forthe test. A Murashige and Skoog salt medium (pH 5.7) containing 0.8%agar was poured into a dual-partitioning sterile petri plate and usedfor a test after being cooled.

The AT-332 (Example 12), Bacillus subtilis GB03 (Comparative Example 12)and Bacillus subtilis MBI600 (Comparative Example 13) were inoculatedrespectively on a sterile paper disc placed on one of the dividedportion of the above petri plate, and germinated seeds of Arabidopsisthaliana were inoculated in the other portion of the petri plate. Theplate inoculated with the bacteria and Arabidopsis thaliana was retainedat 22° C. (12 hours in light/12 hours being cut off from the light) forten days and the plant growth status was observed. The results are shownin photographs (a) to (d) in FIG. 2 including the results of the control(FIG. 2 (a)) where the bacteria were not inoculated. A remarkable effectof promoting plant growth was confirmed with the AT-332 (Example 12;(b)) compared to Bacillus subtilis GB03 (Comparative Example 12; photo(c)) and Bacillus subtilis MBI600 (Comparative Example 13; photo (d)),which are actually sold and used in the United State market.

Example 13 Effect of Promoting Plant Growth of the AT-332 and AT-79Strains (Pot Test)

A pot test was carried out with respect to Chinese cabbage seedlings tomeasure the plant growth promoting effect of the AT-332 and AT-79strains. After culturing the AT-332 and AT-79 strains in a liquid LBmedium for 24 hours, the bacteria cells were collected bycentrifugation. The collected bacteria cells were suspended in a 0.85%sodium chloride aqueous solution so as to be contained at aconcentration of 1×10⁹ CFU/ml. 40 ml of the suspension was mixed per 1kg of the previously sterilized culture soil to serve as the treatedsoil. On the other hand, 40 ml of 0.85% sodium chloride aqueous solutionwas mixed per 1 kg of the previously sterilized culture soil to serve asthe non-treated soil. 100 g of each of the treated soil and non-treatedsoil was put in a plastic pot (70 mm in diameter×68 mm in height)respectively, and seeds of Chinese cabbage (variety: Nozaki ChineseCabbage No. 2) were sowed in the pot. Subsequently, the pots were placedin greenhouse set at 22° C. and the fresh weight of the grown Chinesecabbage was measured after 30 days. The results are shown in FIG. 3. Anexplicit effect of promoting the crop growth of the AT-332 and AT-79strains was confirmed.

What is claimed is:
 1. A method for controlling plant diseases,controlling nematodes, and/or promoting plant growth, said methodcomprising administering, to one or more plants, at least one bacteriaselected from the group consisting of: (a) Bacillus sp. AT-332 strain(deposited under Accession No. NITE BP-1095), containing the 16S rDNAsequence of SEQ ID NO: 2; and (b) Bacillus sp. AT-79 strain (depositedunder Accession No. NITE BP-1094), containing the 16S rDNA sequence ofSEQ ID NO:
 3. 2. A method for controlling plant diseases, controllingnematodes, and/or promoting plant growth, said method comprisingadministering, to one or more plants, a culture from at least onebacteria selected from the group consisting of: (a) Bacillus sp. AT-332strain (deposited under Accession No. NITE BP-1095), containing the 16SrDNA sequence of SEQ ID NO: 2; and (b) Bacillus sp. AT-79 strain(deposited under Accession No. NITE BP-1094), containing the 16S rDNAsequence of SEQ ID NO:
 3. 3. The method of claim 1, wherein the at leastone bacteria is Bacillus sp. AT-79 strain (deposited under Accession No.NITE BP-1094), containing the 16S rDNA sequence of SEQ ID NO:
 3. 4. Themethod of claim 2, wherein the at least one bacteria is Bacillus sp.AT-79 strain (deposited under Accession No. NITE BP-1094), containingthe 16S rDNA sequence of SEQ ID NO:
 3. 5. The method of claim 1, whereinthe at least one bacteria is Bacillus sp. AT-332 strain (deposited underAccession No. NITE BP-1095), containing the 16S rDNA sequence of SEQ IDNO:
 2. 6. The method of claim 2, wherein the at least one bacteria isBacillus sp. AT-332 strain (deposited under Accession No. NITE BP-1095),containing the 16S rDNA sequence of SEQ ID NO:
 2. 7. The method of claim1, wherein said method is a method for controlling plant diseases. 8.The method of claim 1, wherein said method is a method for controllingnematodes.
 9. The method of claim 1, wherein said method is a method forpromoting plant growth.
 10. The method of claim 2, wherein said methodis a method for controlling plant diseases.
 11. The method of claim 2,wherein said method is a method for controlling nematodes.
 12. Themethod of claim 2, wherein said method is a method for promoting plantgrowth.
 13. The method of claim 1, wherein said at least one bacteria isstabilized in a dried form.
 14. The method of claim 7, wherein the oneor more plants to which the at least one bacteria is administered havegray mold disease and/or powdery mildew, and wherein said administeringdecreases the gray mold disease and/or powdery mildew of the one or moreplants.
 15. The method of claim 10, wherein the one or more plants towhich the culture is administered have gray mold disease and/or powderymildew, and wherein said administering decreases the gray mold diseaseand/or powdery mildew of the one or more plants.